Aggressive tumor behavior is usually manifested by increased proliferation, and is most likely associated with "activation" of tumor cells by a number of oncogenes and growth factors. The general goals of this research proposal are to identify markers of tumor proliferation and to investigate their association with clinical outcome in patients with node-negative breast cancer. Specifically, we propose: [1] To investigate the abilities of antibodies against the four proliferation-associated antigens, Ki67, PCNA/cyclin, topoisomerase II, and histone H3, to measure cell-cycle kinetics by immunohistochemistry (IHC) by comparing IHC results to existing flow cytometric S-phase results in a feasibility study of 200 breast carcinomas. [2] To definitively evaluate the prognostic significance of selected proliferation and activation markers using comprehensive "training" and "validation" datasets from over 600 node-negative breast carcinomas. The first anticipated benefit of the proposed studies is the validation of IHC methods to measure tumor proliferation rate. IHC technology possesses several technical and economic advantages which would allow the prognostic evaluation of tumor cell-cell kinetics on a much larger scale than is now possible with current techniques (e.g. flow cytometry). Success in these endeavors will provide clinicians with data regarding proliferation and activation markers that they can apply in making treatment decisions.